The RNA editing ADAR enzymes convert adenosines to inosines in human mRNAs, changing the coding properties of these messages. Thus, ADARs play a pivotal role in the basic process of information transfer during human protein expression. Moreover, proteins translated from edited messages have been implicated in a number of neurodegenerative, psychiatric and behavioral disorders such as stroke, epilepsy, Parkinson's disease, schizophrenia and episodic ataxia. Indeed, deletion of genes encoding ADARs leads to significant behavioral defects in model organisms. However, our understanding of the molecular basis for the fundamental steps in the editing reaction is limited. For instance, why certain adenosines in a substrate mRNA are selectively deaminated continues to be an important and challenging question. In addition, little information is available on regulation of ADAR editing activity. Furthermore, pharmacological methods for controlling ADAR activity do not currently exist. In this competitive renewal of an R01 project, we will address these issues through the application of synthetic chemistry coupled with techniques from biological chemistry. The results of these studies will extend our basic understanding of the process of RNA editing and will lead to new methods for its control. The research proposed here has the following specific aims: 1) We will define the importance of ADAR2 structural features in site-selective RNA editing. This will be accomplished using a novel functional screen in yeast to link changes in ADAR structure to changes in RNA editing efficiency. In addition, the role of ADAR2's identified functional domains (dsRBMs and deaminase domain) will be determined. This will be accomplished through the analysis of trapped complexes using hydroxyl radical probing, nucleotide analog interference, nearest neighbor crosslinking and x-ray crystallography. 2) We will define pathways for regulation of ADAR2's RNA editing activity including by protein kinases, by the binding of inositol hexaphosphate and through the development of artificial regulators that either bind ADAR2 or its RNA substrate. 3) We will generate new nucleoside analogs and modified RNAs to probe different aspects of the ADAR reaction. The design of these analogs is guided by the available crystal structure of the ADAR2 catalytic domain and substrate analog reactivity data.